Neisseria gonorrhoeae is a strict human pathogen that causes greater than 100 million cases worldwide of the sexually transmitted disease gonorrhea each year. Without an effective vaccine, antibiotic therapy remains the principle mechanism to reduce or block the transmission of the gonococcus in the community. Unfortunately, because of the development of mutations or the acquisition of resistance determinants, the gonococcus has developed resistance to relatively inexpensive antibiotics; such resistance now requires the use of more expensive antibiotics that are not always available in poor countries. Multidrug efflux pumps possessed by gonococci have an important role in the ability of gonococci to resist antibiotics and antimicrobials of the innate host defensive system. In particular, our work has shown that the MtrC-MtrD- MtrE efflux pump participates in gonococcal resistance to penicillin and macrolides. This efflux pump also recognizes host antimicrobials, such as antimicrobial peptides and progesterone. We, have shown that the pump is required for gonococci to cause a sustained infection in experimentally-infected female mice and the degree of in vivo fitness is linked to transcriptional regulators that control mtrCDE gene expression. The importance of DNA-binding proteins in regulating gonococcal resistance to antimicrobials is the topic of this R37 MERIT Extension request. In Specific Aim 1 we will define the transcriptional control processes that modulate expression of the mtrR gene, which encodes a repressor of the mirCDE efflux pump operon and can control the expression of numerous genes outside of the mtr locus. We will assess the importance of such regulation in the ability of gonococci to resist antimicrobials and during infection in experimentally infected mice. In Specific Aim 2 we will define the use, regulation and biologic importance of distinct promoters for transcription of the mtrCDE operon. In-Specific Aim 3 we will define the mechanism by which the products of genes controlled by MtrR and other regulatory proteins impact levels of gonococcal susceptibility to antibiotics and host-derived antimicrobials. The results from the proposed work will advance our knowledge regarding how gonococci and other human bacterial pathogens use drug efflux pumps and transcriptional regulatory systems to resist both classical antibiotics as well as mediators of innate immunity